CTLA-4 +49 A/G Polymorphism and the Risk of Lung Cancer: a Meta-analysis

Background and objective Lung cancer is one of the malignant tumors. Gene mutations associated with cellular immune function and regulating the activation and proliferation of immune cells. Several publications have explored the relationship between cytotoxic T lymphocyte antigen-4 (CTLA-4) +49 adenine (A)/guanine (G) polymorphism and susceptibility of lung cancer, but the results remain controversial. Thus, we performed this meta-analysis to derive a more comprehensive estimation of the relationship. Methods All articles addressed lung cancer and polymorphisms of CTLA-4 were searched from the PubMed, EMBASE databases published up to June 29, 2019. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association. Publication bias of relevant studies was examined via Begg's test and funnel plots. Results The meta-analysis included 8 case-control studies covering 4, 430 lung cancer patients and 5, 198 healthy controls from September 2008 to April 2020. The overall eligible data indicated that CTLA-4 +49A/G polymorphisms did not correlate with the elevated lung cancer risk in all genetic comparison models (dominant model: OR=1.037, 95%CI: 0.925-1.161; recessive model: OR=0.968, 95%CI: 0.888-1.055; allele model: OR=0.992, 95%CI: 0.933-1.054; homozygous model: OR=0.980, 95%CI: 0.857-1.121; heterozygous model: OR=1.023, 95%CI: 0.906-1.154). In further stratified analyses, CTLA-4 +49A/G polymorphism was found to be significantly associated with susceptibility to NSCLC in these models (dominant model: OR=1.404, 95%CI: 1.074-1.836; allele model: OR=1.273, 95%CI: 1.034-1.565; homozygous model: OR=1.553, 95%CI: 1.044-2.310; heterozygous model: OR=1.308, 95%CI: 1.062-1.611). Conclusion CTLA-4 +49A/G polymorphism were not associated with the risk of lung cancer but might be a risk factor only in NSCLC.

binding of CTLA-4 and B7 can inhibit the binding of B7 and CD28, interrupt the activation of T cells and participate in the negative regulation of immune response [13] . Many studies have shown that CTLA-4 inhibits T cell proliferation, induces activated T cell apoptosis and controls Treg [14] . In addition, inhibitors targeting CTLA-4 can block the binding of CTLA-4 to B7, inhibit the production of T cell suppression signals and enhance specific anti-tumor immune response. Therefore, the gene status of CTLA-4 may be related to the occurrence of cancer which is a combination of genetic susceptibility and external factors [10] .
There are more than 100 single nucleotide polymorphisms in CTLA-4 gene, such as + 49 adenine (A)/guanine (G), -318 cytosine (C)/thymine (T), -1611 G/A, -1722 T/C, 10223 G/ T polymorphisms, etc [12,15,16] . Among the cancer susceptibility studies, CTLA-4 +49 A/G polymorphism is the most widely studied mutation point. Some studies have shown that CTLA-4 +49 A/G polymorphism is associated with the risk of various cancers, such as breast cancer [17,18] and cervical cancer [19] . However, the relationship between CTLA-4+49 A/G polymorphism and lung cancer is still unclear. Several studies have draw n contradictor y conclusions [10, 2 0 -2 6] . Considering the importance of CTLA-4 in tumorigenesis a nd t he l i m itat ions of si ng le st udy, we conduc ted a comprehensive meta-analysis of published studies to derive a more precise and objective estimation of the relationship between CTLA-4 +49A/G polymorphism and the risk lung cancer.

Identification and eligibility of relevant studies
A s y stemat ic l iterat u re sea rch wa s conduc ted on PubMed, Embase, Wanfang, China National Knowledge Infrastructure (CNKI) and Web of Science databases until June 29, 2019, using the following key words and search strategies: ("CTLA-4" or "cytotoxic T-lymphocyte-associated antigen 4" or "+49 A/G" or "rs231775") and ("polymorphisms" or "mut at ion" or "v a r ia nt s") a nd (" lu ng c a ncer" or "pulmonary cancer" or "lung neoplasms" or "non-small cell lung cancer (NSCLC)" or "NSCLC"). These terms were arranged into different combinations used for search. Only published studies written in English with available full-text were included in this meta-analysis.
The selection criteria were as follows: (1) studies involved the association between CTLA-4 +49 A/G polymorphism and the risk of lung cancer; (2) studies designed as a casecontrolled study; (3) contained available data on the frequency of genotypes including odds ratios (ORs) and 95% confidence intervals (CIs). In addition, the studies that did not meet the inclusion criteria were excluded. Data for the meta-analysis were available from 8 articles including 9 casecontrolled studies (Fig 1).

Data extraction
Two investigators ex tracted data from the eligible studies independently according to the inclusion criteria. When confronted with conf lict, an agreement was settled by discussion with the third reviewer. For each study, the extracted information included: the first author's surname, year of publication, country of origin, ethnicity, sample size of case and control groups, source of controls, histological types, genotyping methods, genotype distributions and Hardy-Weinberg equilibrium (HWE) test. Ethnicities were categorized as Asians or Caucasians.

Statistical analysis
The pooled OR s and its 95%CIs were calculated to

The characteristics of published studies
Following the selected criteria, a total of 105 studies were initially identified through a primary search of PubMed, Embase, Wanfang, CNKI and Web of Science databases and reference lists. Among the studies, 8 full-text articles, including 9 case-control studies harbouring a total of 4,430 cases with lung cancer and 5,198 controls met the inclusion criteria and were included in the meta-analysis for further evaluation, which were accrued between September 2008 and April 2020. Besides, the distribution of genotypes among the controls was consistent under H W E . The f lowchart of literature search and selection procedure is shown in Fig 1. The baseline characteristics of the studies and the distribution of genoty pe are comprehensively listed in Tab 1 and Tab 2. Among the included studies, 6 were based on Asian populations and 3 on Caucasian populations. Genotyping was performed using polymerase chain reaction -restricted fragment length polymorphisms (PCR-R FLP) in 6 st ud ies, TaqMan in 2 st ud ies and single nucleotide polymorphism (SNP) scan kit in 1 study.

Quantitative synthesis results
Overall, the strength of association between CTLA-4 +49 A/G genetic polymorphism and lung cancer risk was evaluated using the pooled ORs and 95%CIs based on five genetic comparison models. A summary of the meta-analysis results for the 9 studied CTLA-4 polymorphism and lung cancer susceptibility is provided in Tab 3.

CTLA-4 +49 A/G and Lung Cancer Risk
In the present meta-analysis, the combined results of all analyses showed that the pooled OR of nine studies was 1 . In addition, when the studies were stratified by ethnicity, source of controls and sample size, no significant differences were found in all genetic models.

Test of heterogeneity
For CT L A-4 + 49 A /G, sig n i f ic a nt heterogeneit y was observed after data were pooled (dominant model: P for heterogeneit y= 0.0 0 0, I 2 =84.4%; Tab 3). In the stratified analyses based on ethnicity, the heterogeneity disappeared in Caucasians group (dominant model: P for heterogeneity=0.065, I 2 =63.5%; Tab 3). When the subjects were stratified on histological type, the heterogeneity disappeared among w ith population-based small cell lung cancer (dominant model: P for heterogeneity=0.177, I 2 =45.1%; Tab 3).

Publication bias
Funnel plot and Begg's test were utilized to evaluate the potential publication biases of the studies involved in the meta-analysis. As shown in Fig 3, the shapes of funnel plots showed no evidence of publication bias in the model. Moreover, Begg's test provided further statistical evidence for the absence of publication bias, indicating that the results of the meta-analysis were reliable.

Discussion
As one of the important immunoglobulin superfamily genes, CTLA-4 is always involved in the development and progression of multiple cancers. In recent years, the study of CTLA-4 polymorphisms involved in the tumorigenesis www.lungca.org increased rapidly due to interest in genetic susceptibility to cancer [10] . Polymorphism in the CTLA-4 gene may confer predisposition to cancer. So far, many previous studies have been carried out to investigate the relationship between +49 A/G polymorphism in CTLA-4 gene and the risk of cancer; however, the data have indicated conflicting results.
The meta-analysis was performed to evaluate more precise results.
In this meta-analysis, a total of 9 eligible and original case-control studies, examined the associations of CTLA-4 +49 A/G polymorphism and lung cancer risk. Our results indicated that +49 A/G polymorphism did not appear  CT L A-4 ac t s a s a ne gat i ve re g u l ator of T-cel l prol i ferat ion a nd ac t iv at ion t h roug h i nduc i ng Fa sindependent apoptosis of activated T cells, retarding T cells at G 1 phase in cell cycle and reducing both interleukin (IL)-2 and IL-2 receptor productions [27,28] . Targeting CTLA-4 with a type of monoclonal antibodies in immunotherapy was a prospective therapeutic approach in many types of tumor by enhancing the activation and expansion of antitumor T cell [29,30] . Thus, CTLA-4 may involve in cancer development and progression and exerted an important function in cancer immunosurveillance. The current meta-analysis results showed an increased risk of NSCLC for carriers of the A allele. The +49 A/G polymorphism of CTLA-4 refers to a threonine (Thr) to alanine (Ala) substitution in the leading peptide of CTLA-4 receptor [31] . The studies reported that the 49G allele reduced CTLA-4 production than the 49A allele because of its lower messenger ribonucleic acid (RNA) efficiency and this polymorphism enhances the combination between CTLA-4 protein and its ligand B7.1. Individuals with 49GG genotype may lead to greater T-cell proliferation and stronger binding to ligand B7.1 than that with 49 A A genotype [10] . According to our current results, these findings revealed that the Thr-to-Ala change in CTLA-4 may be related with the risk of NSCLC.
The heterogeneity and publication bias are of importance which may affect the results of meta-analysis. Significant heterogeneity existed in overall comparisons in the dominant www.lungca.org model. After subgroup analyses by ethnicity, histological type, source of controls and sample size, the heterogeneity effectively was decreased or almost removed in some subgroups, suggesting different gene-environment factors effect on different histological type or different population. In the meta-analysis, publication bias was analyzed by Begg's funnel plots and the Egger's test and no significant publication bias was detected, suggesting the reliability of our results.
In addition, some limitations should be considered. First, the number of studies was limited, which may affect the power to reveal a reliable association. In the future, large numbers of studies need to be conducted to validate these association. Second, all case-control studies were from Asia and Caucasians, thus our results may be only applicated to these ethnic groups. Third, only published studies were recruited and publication bias might have occurred ineluctably. Fourth, data were not performed to further stratified analysis by other factors, such as environmental and lifestyle factors, because the information extracted from the primary publication was finite. I n conclusion, t he resu lts f rom t he meta-a na lysis demonstrated that CTLA-4 +49 A/G polymorphism was a risk factor for NSCLC. Future more large-scale and well-designed studies with functional evaluations should be carried out to definite the results and investigate the molecular mechanisms of CTLA-4 modify cancer risk.

Author contributions
Hu J conceived and designed the study. Wei ZL analyzed the data. Wei ZL and Zhang SQ contributed analysis tools. Wei ZL and Zhang SQ provided critical inputs on design, analysis, and interpretation of the study. All the authors had access to the data. All authors read and approved the final manuscript as submitted.